Shiga-like Toxin Producing E. Coli Research Articles

Occurrence, pathotypes, and antimicrobial resistance profiles of diarrheagenic Escherichia coli strains in animal source food products from public markets in Mashhad, Iran

Diarrheagenic Escherichia coli (DEC) strains are common pathogens that transmitted through the consumption of contaminated foods, and cause acute intestinal diseases in human. The present study was conducted on 300 samples including raw milk, meat and some of their products from November 2016 to October 2017. Microbiological and molecular diagnostic techniques were used to identification of DEC. A total of 69% (207/300) E. coli was isolated and the frequency of DEC was 25.6% (53/207). The pathogenic groups of DEC recovered from the isolates had the following profile: Shiga-like toxin producing E. coli (STEC): 62.3%, enteropathogenic E. coli (EPEC): 24.5%, enteroaggregative E. coli (EAEC): 9.4%, and enterotoxigenic E. coli (ETEC): 3.8%. Neither enteroinvasive E. coli (EIEC) nor diffusely adherent E. coli (DAEC) were found. The highest incidence values were found in hamburgers (46.7%), and the highest rate of DEC positive prevalence was in March. Of the DEC strains, 90.6% (48/53) displayed resistance to at least one antibiotic and the highest level of resistance was found for tetracycline (69%). The obtained results revealed that the studied animal source food products may easily act as a reservoir of DEC with a potential ability to transfer antibiotic resistance and virulence genes to the gastrointestinal microbiota. Therefore, it is of paramount importance to develop effective strategies for improving food safety and updated guidelines for the prudent use of antimicrobial agents in Iran.

Prevalence of O-serogroups, virulence genes, and F18 antigenic variants in Escherichia coli isolated from weaned piglets with diarrhea in Korea during 2008-2016.

To diagnose colibacillosis, detection of O-serogroups and virulence genes has been recommended worldwide. The prevalence of virulence factors can fluctuate over time. The objectives of this study were to determine the prevalence of O-serogroups, virulence genes, and F18 subtypes among pathogenic Escherichia coli isolated from weaned piglets with diarrhea in Korea. Between 2008 and 2016, 362 E. coli were isolated from weaned piglets with diarrhea. Hemolysis was determined in blood agar, and O-serogroups were identified using the slide agglutination technique. The genes for the toxins and fimbriae were amplified by polymerase chain reaction (PCR). Real-time PCR was conducted to discriminate between F18 subtypes. Although the most prevalent serogroup was O149 (11.3%) in the last 9 years, O139 (19.1%) became the most prevalent in recent years (2015–2016). The most predominant pathotype was enterotoxigenic E. coli (61.3%). The frequencies of Shiga-like toxin-producing E. coli (STEC) (23.4%), O139 (19.1%), Stx2e (35.1%), and F18ab (48.7%) increased over the most recent years. Although enterotoxigenic E. coli was the most predominant pathotype, the frequencies of O139, Stx2e, STEC, and F18ab have increased in recent years. These results demonstrate that there have been temporal changes in the predominant O-serogroups and virulence genes over the last decade in Korea. These findings can be practicable for use in epidemiology and control measures for enteric colibacillosis in Korean piggeries.

Shiga-like Toxin Producing E. coli Identified in Patients with Multiple Myeloma (MM) Hospitalized Due to Acute Onset Diarrhea; First Report on the Clinical Use of the Biofire Film Array Gastrointestinal Panel

Introduction:Infections are a major cause of morbidity and mortality in patients with multiple myeloma (MM) and of these, one of the most frequent is gastroenteritis. This can be explained by immunoparesis, a known manifestation of MM and immunosuppression due to multiple lines of treatment.Other causes of diarrhea such as drugs or gastro-intestinal infiltration by plasma cells should also be excluded.Immunoglobulin A(IgA) is a major immunoglobulin on mucosal surface and defends the bowel from pathogens invasion. Data exists in literature about the protective role of IgA against Shiga toxin.Early isolation of pathogen and correct therapeutic intervention are important and can be lifesaving for MM myeloma patients.Until recently, diagnostic clinical laboratories utilized different methodologies to detect bacterial, parasitic, and viral causes of gastroenteritis. These tests have a number of disadvantages, including poor sensitivity, potentially long turnaround times, and complicated workflows. In addition, there are limited or almost no testing methods routinely available for most strains of Escherichia coli causing diarrhea.The Film Array GI Panel, is a relatively new method introduced recently, which provides comprehensive, simultaneous detection of 22 different enteric pathogens on stool specimens with a turnaround time as short as 1 hour . Because of its high cost it is not used routinely in all patients presenting to the emergency room with acute diarrhea.Here we summarize our real-life experience, using the BioFire PCR-Arrayfor 4 patients with relapsed /refractory MM who were admitted to our medical center with an acute presentation of gastroenteritisMethods:The Infectious Diseases Unit at our medical centre decided to introduce a strategic approach of the use of BioFire PCR-Array for the examination of stool samples, in immunocompromised patients who present to the emergency room with acute diarrhea.The panel used includes the following 22 pathogens: Campylobacter spp., Clostridium difficile(toxin A/B), Plesiomonas shigelloides, Salmonella spp., Vibriospp., Vibrio cholerae, Yersinia enterocolitica, enteroaggregative E. coli, enteropathogenic E. coli, enterotoxigenic E. coli, Shiga-like toxin-producing E. coli (stx1 and stx2) (including specific detection of E. coli O157), Shigella spp./enteroinvasive E. coli, Cryptosporidium spp., Cyclospora cayetanensis, Entamoeba histolytica, Giardia lamblia, adenovirus F 40/41, astrovirus, norovirus GI/GII, rotavirus A, and sapovirus.The assay is sent on the first day of patient admission, and results are reported to the Infectious Diseases Unit within one hour.Here we summarize the results obtained in four patients with MM treated at the Hemato-Oncology Unit in Bnai Zion Medical Centre , Haifa , who were hospitalized in 2018 due to acute diarrhea and had positive PCR-Biofire results;Results:All patients examined had relapsed/refractory MM and had received 2-5 treatment lines for MM.Two of the four patients had neutropenia on admission, two had low IgG level, three had low IgM level and all four had low IgA levels. Of the four patients with MM and acute diarrhea, evaluated by BioFire Film Array, one had two episodes of acute diarrhea on different occasions and was evaluated twice.Five pathogens were detected by BioFire FilmArray, STEC (Shiga-like toxin producing E. coli) was the most common bacteria encountered and was detected 3 times: EPEC (Enteropathogenic E. coli) and ETEC (Entertoxogenic E. coli) were detected one time each.Careful review of the literature did not identify any previous case report of patients with multiple myeloma who had Shiga-like toxin producing E. coli.All patients received antibiotics-Azithromycin and improved.Conclusion: In our experience using the PCR- Biofire assay we show that this approach is feasible and can be used safely to tailor therapy in immunocompromised patients. In all cases, E.Coli was found, most frequently STEC (Shiga-like toxin producing E. coli). Using this approach enable us to identify new pathogens causing diarrhea in immunocompromised patients, extremely quickly. These preliminary results reveal the need for more clinical experience using the Biofire FilmArray as a diagnostic test for patients with MM and other haematological malignancies, who present acutely with severe diarrhea. DisclosuresTadmor:NOVARTIS: Consultancy; ABBVIE: Consultancy; JNJ: Consultancy; PFIEZER: Consultancy; ROCHE: Research Funding. Polliack:ABBVIE: Consultancy; ROCHE: Research Funding.

Validation of the Applied Biosystems RapidFinder Shiga Toxin-Producing E. coli (STEC) Detection Workflow.

The Applied Biosystems™ RapidFinder™ STEC Detection Workflow (Thermo Fisher Scientific) is a complete protocol for the rapid qualitative detection of Escherichia coli (E. coli) O157:H7 and the "Big 6" non-O157 Shiga-like toxin-producing E. coli (STEC) serotypes (defined as serogroups: O26, O45, O103, O111, O121, and O145). The RapidFinder STEC Detection Workflow makes use of either the automated preparation of PCR-ready DNA using the Applied Biosystems PrepSEQ™ Nucleic Acid Extraction Kit in conjunction with the Applied Biosystems MagMAX™ Express 96-well magnetic particle processor or the Applied Biosystems PrepSEQ Rapid Spin kit for manual preparation of PCR-ready DNA. Two separate assays comprise the RapidFinder STEC Detection Workflow, the Applied Biosystems RapidFinder STEC Screening Assay and the Applied Biosystems RapidFinder STEC Confirmation Assay. The RapidFinder STEC Screening Assay includes primers and probes to detect the presence of stx1 (Shiga toxin 1), stx2 (Shiga toxin 2), eae (intimin), and E. coli O157 gene targets. The RapidFinder STEC Confirmation Assay includes primers and probes for the "Big 6" non-O157 STEC and E. coli O157:H7. The use of these two assays in tandem allows a user to detect accurately the presence of the "Big 6" STECs and E. coli O157:H7. The performance of the RapidFinder STEC Detection Workflow was evaluated in a method comparison study, in inclusivity and exclusivity studies, and in a robustness evaluation. The assays were compared to the U.S. Department of Agriculture (USDA), Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook (MLG) 5.09: Detection, Isolation and Identification of Escherichia coli O157:H7 from Meat Products and Carcass and Environmental Sponges for raw ground beef (73% lean) and USDA/FSIS-MLG 5B.05: Detection, Isolation and Identification of Escherichia coli non-O157:H7 from Meat Products and Carcass and Environmental Sponges for raw beef trim. No statistically significant differences were observed between the reference method and the individual or combined kits forming the candidate assay using either of the DNA preparation kits (manual or automated extraction). For the inclusivity and exclusivity evaluation, the RapidFinder STEC Detection Workflow, comprising both RapidFinder STEC screening and confirmation kits, correctly identified all 50 target organism isolates and correctly excluded all 30 nontarget strains for both of the assays evaluated. The results of these studies demonstrate the sensitivity and selectivity of the RapidFinder STEC Detection Workflow for the detection of E. coli O157:H7 and the "Big 6" STEC serotypes in both raw ground beef and beef trim. The robustness testing demonstrated that minor variations in the method parameters did not impact the accuracy of the assay and highlighted the importance of following the correct incubation temperatures.

Persistence of Pathogenic and Non-Pathogenic Escherichia coli Strains in Various Tropical Agricultural Soils of India.

The persistence of Shiga-like toxin producing E. coli (STEC) strains in the agricultural soil creates serious threat to human health through fresh vegetables growing on them. However, the survival of STEC strains in Indian tropical soils is not yet understood thoroughly. Additionally how the survival of STEC strain in soil diverges with non-pathogenic and genetically modified E. coli strains is also not yet assessed. Hence in the present study, the survival pattern of STEC strain (O157-TNAU) was compared with non-pathogenic (MTCC433) and genetically modified (DH5α) strains on different tropical agricultural soils and on a vegetable growing medium, cocopeat under controlled condition. The survival pattern clearly discriminated DH5α from MTCC433 and O157-TNAU, which had shorter life (40 days) than those compared (60 days). Similarly, among the soils assessed, the red laterite and tropical latosol supported longer survival of O157-TNAU and MTCC433 as compared to wetland and black cotton soils. In cocopeat, O157 recorded significantly longer survival than other two strains. The survival data were successfully analyzed using Double-Weibull model and the modeling parameters were correlated with soil physico-chemical and biological properties using principal component analysis (PCA). The PCA of all the three strains revealed that pH, microbial biomass carbon, dehydrogenase activity and available N and P contents of the soil decided the survival of E. coli strains in those soils and cocopeat. The present research work suggests that the survival of O157 differs in tropical Indian soils due to varied physico-chemical and biological properties and the survival is much shorter than those reported in temperate soils. As the survival pattern of non-pathogenic strain, MTCC433 is similar to O157-TNAU in tropical soils, the former can be used as safe model organism for open field studies.

Nowe spojrzenie na bakteryjne patogeny odzwierzęce stanowiące zagrożenie dla człowieka

This article presents the problem of evolutionary changes of zoonotic pathogens responsible for human diseases. Everyone is exposed to the risk of zoonotic infection, particularly employees having direct contact with animals, i.e. veterinarians, breeders, butchers and workers of animal products' processing industry. The article focuses on pathogens monitored by the European Centre for Disease Prevention and Control (ECDC), which has been collecting statistical data on zoonoses from all European Union countries for 19 years and publishing collected data in annual epidemiological reports. Currently, the most important 11 pathogens responsible for causing human zoonotic diseases are being monitored, of which seven are bacteria: Salmonella spp., Campylobacter spp., Listeria monocytogenes, Mycobacterium bovis, Brucella spp., Coxiella burnetti and Verotoxin-producing E. coli (VTEC)/Shiga-like toxin producing E. coli (STEC). As particularly important are considered foodborne pathogens. The article also includes new emerging zoonotic bacteria, which are not currently monitored by ECDC but might pose a serious epidemiological problem in a foreseeable future: Streptococcus iniae, S. suis, S. dysgalactiae and staphylococci: Staphylococcus intermedius, S. pseudintermedius. Those species have just crossed the animal-human interspecies barrier. The exact mechanism of this phenomenon remains unknown, it is connected, however, with genetic variability, capability to survive in changing environment. These abilities derive from DNA rearrangement and horizontal gene transfer between bacterial cells. Substantial increase in the number of scientific publications on this subject, observed over the last few years, illustrates the importance of the problem.

Diagnosis and prevalence of enteropathogenic bacteria in children less than 5 years of age with acute diarrhea in Tehran children's hospitals

Acute diarrhea disease is the second cause of death among all infectious diseases in children younger than 5 years of age worldwide. The aim of this study was to employ a combination of biochemical, microbiological and molecular diagnostic techniques to investigate the stools of Iranian children with acute diarrhea for bacterial enteropathogens. Diarrheagenic Escherichia coli, Shigella spp., Salmonella spp., Campylobacter spp. and Yersinia spp., were investigated from June 2003 to June 2005, in 1087 children less than 5 years old with acute diarrhea. Stool specimens from children were studied for enteropathogens both by standard culturing and molecular methods. This study was designed on hospital based. The highest incidence values were found in the summer and in children less than 1-year-old (42.7%). The Pathogenic bacteria recovered out from fecal samples of 555 (55.1%) patients had the following profile: Shigella spp. (26.7%) was the most prevalent bacterial pathogen and Shiga-like toxin producing E. coli (STEC) and Enteroaggregative E. coli (EAEC) 105 (18.9%) and 92 (16.6%) had the second and third highest prevalence, respectively. Enteropathogenic E. coli (EPEC), Campylobacter, Salmonella and Enterotoxigenic E. coli (ETEC) were found in 70 (12.6%), 60 (10.8%), 42 (7.6%), and 38 (6.8%) positive samples, respectively. In this study neither Yersinia nor E. coli O157:H7 were found. Of the 30 co-infections detected, Shigella flexneri and Campylobacter jejuni accounted for more than 50%. Information about the prevalence of wide-range Shigella and STEC may facilitate the control and management of infant diarrhea diseases in Iran. The results of this study suggest that comprehensive surveys are needed in different parts of the country in order to identify the incidence of different enteropathogenic diarrhea, especially diarrheagenic E. coli in children in Iran.

A case of lethal enteroviral haemolytic uraemic syndrome?

Sir, In the recent years much has been learnt about haemolytic uraemic syndrome (HUS), which is defined by symptomatic or bloody diarrhoea, acute nephropathy, thrombocytopenia and microangiopathic haemolytic anaemia. Often HUS is complicated by neurological symptoms in children [1]. The main aetiological culprit is Shiga-like toxin-producing E. coli (STEC) accounting for ∼85% of HUS cases. Recently the aetiological role of enteroviruses in HUS has been studied and rejected as being an aetiological agent [3] even though earlier case reports suggested a role (see references in [3]). We present a case of HUS, which was probably due to enteroviral infection. A 17-month-old girl, who was previously healthy, was admitted with bloody diarrhoea preceded by vomiting for a day or two. A few hours into the admission the girl developed a seizure and was transferred to an ICU. Extensive clinical and laboratory testing was carried out (Table 1). Tests showed uraemia, thrombocytopenia, haemolytic anaemia, leucocytosis and anuria. The patient developed cardiac arrest at 8 h into the admission. Resuscitation with concurrent peritoneal dialysis was attempted for an hour without success. On autopsy we found a normally developed girl without any deformities. The autopsy revealed a thickened haemorrhagic colon, stasis and oedema in the lungs, hydrothorax and hydroperitoneum. The CNS was without lesions (vascular or any other). There were no signs of iatrogenic injury. On histopathological examination we found signs of endothelial damage in the small vessels of the kidneys (Figure 1) and the intestine (with thrombosis), fragments of thrombi in the small lung vessels and a necrotic colon with submucosal haemorrhage and leukocyte infiltration. No thrombi were found in the brain. Concurrent to the autopsy we requested several tests expecting to find a STEC infection but instead we found an enteroviral infection (Table 1). Our case indicates that enteroviral infections may be able to inflict HUS, and contradicts other studies of HUS [3]. It is clear from the above-mentioned findings that the case presented as HUS, with all of its cardinal findings, uraemia, thrombocytopenia, haemolytic anaemia, and endothelial damage in the kidney and intestine. We used stool and tissue cultures for the bacterial tests whereas viral tests included PCR, serology and cultures. Sadly subtyping (using cell cultures) was unsuccessful after several attempts. A weakness in our report is that we do not have multiple tests (i.e. serology) to exclude a STEC infection, only a standard stool culture. Furthermore, one could speculate that the finding of enteroviral RNA is coincidental or from “leakage” of colonic virus into blood. In support of our conclusion, enteroviruses are known to be able to infect colonic cells and endothelial cells, and damage both by either lytic or immunological mechanisms, the prerequisites for inducing HUS [4,5]. Furthermore our detection techniques are more sensitive [6,7] than the ones used earlier in studies [3]. Our case underlines the need for a large-scale epidemiological investigation of STEC-negative HUS cases using the most recent sensitive diagnostic tests especially considering the poorer prognosis of STEC-negative cases [3]. Conflict of interest statement. None declared.

Clonal Diversity of Escherichia Coli Isolates from Marketed Beef in East Malaysia

Escherichia coli, including Shiga-like toxin producing E. coli (STEC), serogroup O157:H7 and E. coli O157, were isolated from raw beef marketed in Sarawak and Sabah, East Malaysia. Molecular subtyping by pulsed-field gel electrophoresis (PFGE) was performed on 51 confirmed E. coli isolates. Of the 51 isolates, five were E. coli O157:H7, four E. coli O157, two non-O157 STEC and 40 other E. coli isolates (non-STEC). Digestion of chromosomal DNA from these E. coli isolates with restriction endonuclease XbaI (5′-TCTAGA-3′), followed by PFGE, produced 45 restriction endonuclease digestion profiles (REDPs) of 10–18 bands. E. coli O157:H7 isolates from one beef sample were found to have identical PFGE profiles. In contrast, E. coli serogroup O157 from different beef samples displayed considerable differences in their PFGE profiles. These suggested that E. coli isolates of both serogroups were not closely related. A large variety of PFGE patterns among non-STEC isolates were observed, demonstrating a high clonal diversity of E. coli in the beef marketed in East Malaysia. The distance matrix values (D), calculated showed that none of the pathogenic E. coli strains displayed close genetic relationship with the non-STEC strains. Based on the PFGE profiles, a dendrogram was generated and the isolates were grouped into five PFGE clusters (A–E). From the dendrogram, the most related isolates were E. coli O157:H7, grouped within cluster B. The STEC O157:H7 beef isolates were more closely related to the clinical E. coli O157:H7 isolate than the E. coli O157:H7 reference culture, EDL933. Cluster A, comprising many of other E. coli isolates was shown to be the most heterogeneous. PFGE was shown to possess high discriminatory power in typing pathogenic and non-pathogenic E. coli strains, and useful in studying possible clonal relationship among strains.

Gene encoding virulence markers among <i>Escherichia coli</i> isolates from diarrhoeic stool samples and river sources in rural Venda communities of South Africa

River water sources and diarrhoeic stools of residents in the Venda Region, Limpopo Province of South Africa were analysed for the prevalence of Escherichia coli (E. coli) and the presence of virulence genes among the isolates. A control group of 100 nondiarrhoeic stool samples was included. Escherichia coli was isolated and identified by standard cultural and biochemical methods. Pathogenicity of environmental and human isolates was determined by amplification of genes associated with virulence of E. coli, using specific primers. Of a total of 228 water and river sediment samples screened, E. coli was recovered from 200 (87.7%), and 135 (67.5%) of these had one or more genes associated with pathogenicity. The highest frequency of isolation of pathogenic strains was found in Ritavi River water and sediment (80.6%), followed by Lotanyanda River (76.9%), and the least (45.8%) in Nzhelele River 2. Escherichia coli was recovered from all of the 252 diarrhoeic stools tested (100%), and 119 (47.28%) of these had one or more genes associated with pathogenicity. The frequency of isolation of potential pathogenic E. coli from humans was highly significant (t = 6.3; pdย•0.01) in comparison to water isolates. Cytotoxic necrotizing Factor 1 (cnf1) and cytotoxic necrotising Factor 2 (cnf2) coding for necrotoxigenic E. coli (NEC); bundle-forming pilus (bfpA) and enteropathogenic attachment and effacement (eaeA) coding for enteropathogenic E. coli (EPEC), occurred in 35% and 34% respectively of river isolates. Heat-stable (ST) and heat-labile (LT) toxin genes coding for enterotoxigenic (ETEC) and Shiga-like toxin 1 (Stx1) and Shiga-like toxin 2 (Stx2) coding for Shiga-like toxin-producing E. coli (STEC) were not encountered in the river isolates. Isolates from stool samples had 21.8% and 12.6% of EPEC and NEC strains respectively; while enterotoxigenic (ETEC), Shiga-like toxin-producing (STEC) and enteroaggregative E. coli (EAEC) had a prevalence of 5%, 5.8% and 5.8% respectively. One human isolate possessed stx2 and eaeA indicating E. coliO157: H7. No genes associated with pathogenicity were observed in human non-diarrhoeic stool isolates. Results have revealed a possibility of a recycling of pathogenic E. coli strains, particularly the EPEC and NEC strains, between the water sources and the local population. Water SA Vol.30(1): 37-42

Escherichia Coli O157 H7 and Shiga-like-toxin- producing Escherichia Coli in China.

Escherichia coli (E. coli) is one of the facultative anaerobes of the human intestinal tract, usually harmless. Infections due to pathogenic E.coli may result in urinary tract infections, sepsis, meningitis and enteric disease. Diarrheagenic E.coli has been classified into several categories, such as enterotoxigenic E.coli (ETEC), entero-invasive E.coli (EIEC), entero-pathogenic E.coli (EPEC), enteroaggregative E.coli (EaggEC) and entero-hemorrhagic E.coli (EHEC). A variety of virulence or potential virulence factors for diarrheagenic E.coli have been identified. The nomenclature of diarrheagenic E.coli is based on virulence factors. In 1977 Konowalchuk et al reported that some strains of pathogenic E.coli O26 : H11 produced a toxin with a profound cytopathic effect on Vero cells, and named it verotoxin (VT). O’Brien et al noted that the VT reported by Konowalchuk et al was strikingly similar to Shiga toxin (Stx) produced by-Shigella dysenteriae type 1, and it could be neutralized by antiStx, thus a new nomenclature, Shiga-like toxin (SLT), appeared. An alternative nomenclature is “Shiga toxin” (ST), which indicated t hat the specific cytotoxin described by Konowalchuk et al is esse ntially identical at the genetic and protein levels with the Stx produced by S. dysenteriae I discovered some 100 years ago. Consequently, SLT, ST and VT have been used interchangeably, resulting in the name of verotoxinproducing E.coli (VTEC), shiga-like-toxinproducing E.coli (SLTEC) and shiga-toxin-producing E.coli (STEC) coexisted in literature. However, it must be noted that E.coli O157 : H7 is the main serotype of EHEC recognized at present. Hemorrhagiccolitis (HC) and hemolytic uremic syndrome (HUS) are life threatening, which are often caused by STEC or EHEC. So far as the diseases are concerned, E.coli O157 : H7 should belong to EHEC. All of the EHEC strains are believed to be pathogenic. As for its toxin, E.coli O157 : H7 should belong to VTEC, SLTEC or STEC. However, not all of the STEC strains could cause HC or HUS. The confusion from the nomenclature may be clarified in future when the pathogenic mechanisms of bacteria are fully understood. Food-borne outbreaks of SLTEC disease appear to be increasing in the world. Mass-produced and mass-distributed food can involve large numbers of people in short time. SLTEC strains belong to a very diverse range of serotypes, among which O157 : H7 is most commonly associated with large outbreaks. In the summer of 1996 in Japan, a largest outbreak in the world caused by E.coli O157 : H7 was reported, in which about 10 000 cases were identified. Chinese grovernment and society became aware of the importance of E.coli O157 : H7 from the Japanese outbreak. An informal national network for detection of E.coli O157 : H7 was organized in April 1997, involving about 30 public health laboratories from different provinces and municipalities.

Molecular epidemiology of Escherichia coli O157:H7 by restriction fragment length polymorphism using Shiga-like toxin genes.

The purpose of this study was to assess a simplified method for interstrain differentiation of Escherichia coli O157:H7 and other Shiga-like toxin-producing E. coli (SLTEC) strains. A method based on the use of nucleic acid probes from Shiga-like toxin (SLT) I and II structural genes was used to generate restriction fragment length polymorphism (RFLP) patterns of SLTEC strains, (SLT-RFLP patterns) resulting from digestion of isolated genomic DNA with four different restriction enzymes (BamHI, EcoRI, HindIII, and PvuII) used separately. A total of 165 SLTEC strains from clinical, food, and environmental sources, including O157:H7 isolates from four food-borne outbreaks in Canada and the United States, were analyzed in the study. SLT-RFLP demonstrated that E. coli O157:H7 strains from each food-borne outbreak had the same unique SLT-RFLP pattern. Fifty-two SLT-RFLP types were found among 96 E. coli O157:H7 isolates from sporadic cases of hemorrhagic colitis and hemolytic uremic syndrome in Washington state. The use of the SLT probes proved to be a very powerful method for interstrain differentiation of SLTEC strains. Although the use of each of the enzymes alone did not give enough differentiative power to be used in epidemiological studies, the combination of patterns generated by two restriction enzymes (EcoRI and PvuII, used separately) provided the desired sensitivity for such studies. The results clearly demonstrate the usefulness of the method for studying the molecular epidemiology of E. coli O157:H7. The method is also suitable for establishing an epidemiological database, in terms of both sensitivity and ease of compilation and interpretation of results.

Neutralizing antibodies against Shiga-like toxins from Escherichia coli in colostra and sera of cattle

Previous of present infection with Shiga-like toxin producing E. coli (SLTEC) was detected by an indirect neutralization assay of antibody titer. Bovine colostra and sera blocked the cytotoxic effects of Shiga-like toxin on Vero cell monolayers. SLT neutralizing antibodies were present in 84.0% ( 189 225 ) of the colostrum samples from randomly chosen cows in Bavaria, Germany. While all of the colostra with neutralizing activity reacted with SLT-I, only 14.7% neutralized both SLT-I and - II. Approximately 93.0% ( 37 40 ) of sera from heifers had SLT neutralizing activity. To quantify the neutralizing antibodies, colostra were tested in the Vero cell assay for their capability to reduce the 50% cytotoxic dose (CD 50) of SLT standards, where the neutralizing units/ml (nu/ml) equal the log 10 of CD 50 reduction. Almost half of reactive colostra (48.7%) reduced the CD 50 of the SLT-I standard by 10 4 to 10 5 (4–5 nu/ml). Higher reactivity (5–7 nu/ml) was found in 46.5% of positive colostra. The remaining colostra samples had over 7 nu/ml. To determine if the colostra were blocking receptors for SLT on Vero cells, cells were preincubated with colostra, and SLT was later added. No neutralizing activity was detected, indicating the reactivity of colostra was directed against SLT. When the colostra were subjected to ammonium sulphate precipitation and DEAE anion exchange chromatography, high levels of neutralizing activity were found in the IgG 1 containing fractions. Colostrum fractions were tested for SLT-I binding antibodies in a capture ELISA, based on the binding of SLT-I to the toxin receptor analogue P 1-glycoprotein. Only fractions from colostra with over 5 nu/ml were reactive in this assay, indicating the ELISA was less sensitive than the Vero cell assay. The results support the theory that SLTEC exposure of cows in Germany is more widespread than expected from epidemiological studies based on bacterial isolation. This possibly indicates a higher risk of human SLTEC infection via beef and milk products.

A Prospective Study of Shiga‐Like Toxin‐Associated Diarrhea in a Pediatric Population

SummaryAlthough population‐based studies have shown that children have the highest age‐specific incidence of infection with the Shiga‐like toxin‐producing E. coli (SLTEC), these sporadic case series were not focused specifically on the pediatric age group. We undertook a prospective study to determine the frequency of detection of SLT in an exclusively pediatric population. The study design minimized ascertainment and referral bias by systematically defining the population by the presence of diarrheal symptoms rather than by specific diagnosis, previous submission of stool for culture, or referral to a diarrhea study. All children < 10 years of age hospitalized at a tertiary care pediatric hospital, irrespective of admission diagnosis, were surveyed prospectively at admission and for 2 days thereafter for the presence of defined diarrheal symptoms. From May 1, 1991, to April 30, 1992, 227 patients and 92 age‐ and season‐matched controls were enrolled. Fecal SLT was detected in six (2.6%) patients, three of whom had E. coli 0157:H7 organisms were isolated; SLT was not found in any of the controls. SLT was more commonly detected in children 2–10 years of age and in bloody stools. Salmonella was isolated in six (2.6%) cases, Shigella in five (2.2%), and Yersinia in three (1.3%); rotavirus was detected in 46 (20.3%). Two patients with SLT‐associated diarrhea had hemolytic uremic syndrome (HUS), and four had hemorrhagic colitis. SLT‐associated diarrhea occurred in the summer and fall months in contradistinction to that with rotavirus, which occurred in the winter and spring. Because enteric infection with SLTEC may have serious sequelae, such as HUS, and because it occurs with a frequency comparable to that of other bacterial entero‐pathogens, the evaluation of diarrhea in pediatric patients should include a search for SLTEC, particularly E. coli 0157:H7.

A Prospective Study of Shiga-Like Toxin-Associated Diarrhea in a Pediatric Population

Although population-based studies have shown that children have the highest age-specific incidence of infection with the Shiga-like toxin-producing E. coli (SLTEC), these sporadic case series were not focused specifically on the pediatric age group. We undertook a prospective study to determine the frequency of detection of SLT in an exclusively pediatric population. The study design minimized ascertainment and referral bias by systematically defining the population by the presence of diarrheal symptoms rather than by specific diagnosis, previous submission of stool for culture, or referral to a diarrhea study. All children < 10 years of age hospitalized at a tertiary care pediatric hospital, irrespective of admission diagnosis, were surveyed prospectively at admission and for 2 days thereafter for the presence of defined diarrheal symptoms. From May 1, 1991, to April 30, 1992, 227 patients and 92 age- and season-matched controls were enrolled. Fecal SLT was detected in six (2.6%) patients, three of whom had E. coli O157:H7 organisms were isolated; SLT was not found in any of the controls. SLT was more commonly detected in children 2-10 years of age and in bloody stools. Salmonella was isolated in six (2.6%) cases, Shigella in five (2.2%), and Yersinia in three (1.3%); rotavirus was detected in 46 (20.3%). Two patients with SLT-associated diarrhea had hemolytic uremic syndrome (HUS), and four had hemorrhagic colitis. SLT-associated diarrhea occurred in the summer and fall months in contradistinction to that with rotavirus, which occurred in the winter and spring.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of Escherichia coli serotype O157:H7 and other Shiga-like-toxin-producing E. coli from dairy cattle

We examined 1,266 fecal specimens from healthy cattle during the investigations of two sporadic cases of hemolytic uremic syndrome associated with raw milk consumption and an outbreak of gastroenteritis and hemolytic uremic syndrome caused by Escherichia coli serotype O157:H7. We collected specimens from heifers, calves, and adult cows on 22 farms, in a stockyard, and in a packing house. We also collected 3 raw hamburger specimens from a restaurant and 23 raw milk samples from two farms. All specimens were examined for E. coli O157:H7 by using sorbitol-MacConkey agar, H immobilization, O157 agglutination, and tissue culture cytotoxicity. E. coli O157:H7 was isolated from 16 heifers or calves and 1 adult cow on 22 farms, 1 stockyard calf, 2 beef specimens, and 1 raw milk sample. Selected fecal specimens were also examined for the presence of other Shiga-like-toxin-producing E. coli (SLTEC) by testing polymyxin B extracts of colony sweeps and then testing individual colonies for toxin production. SLTEC other than O157 was isolated from 8 of 10 farms investigated and from the stockyard; 8% of adult cows and 19% of heifers and calves were positive for SLTEC. Several animals were positive for SLTEC by colony sweep only. This investigation demonstrates that dairy cattle are a reservoir of E. coli O157:H7 and other SLTEC.